The standard of care for many types of cancer, including malignant tumors of the breast2-4 and thyroid5, 6, is resection of the tumor and surrounding tissue. Excised tissue is evaluated postoperatively using conventional histology; however this requires a delay of up to one day for processing, embedding, sectioning and staining7. This delay between surgery and pathological assessment poses complications when surgical treatment depends on pathology findings. For example, breast-conserving therapy and radiation is a standard of care in early breast cancer because it achieves survival and local recurrence rates equivalent to those of mastectomy while providing improved post-operative quality of life8-12. Unfortunately because of the limited sensitivity of gross examination, residua tumor is present near the surgical margin in up to 40% of cases16, 17 and patients therefore often require repeat surgeries. Similarly, in the resection of papillary thyroid neoplasm, a findin of vascular or capsular invasion following a lobectomy typically requires a second surgery to perform a total or near-total thyroidectomy6. Therefore, there is a need for imaging technology to assess cancer pathology in real-time. A potential alternative to conventional histopathology, two photon microscopy (TPM) of surgical specimens retains the high resolution and molecular specificity of conventional histopathology using endogenous or exogenous contrast25, 26, and 37. Fluorescent lifetime imaging microscopy (FLIM) is a related imaging modality that can be implemented using TPM29. TPM with fluorescence lifetime imaging (TPM-FLIM) provides significantly greater molecular contrast then conventional TPM by measuring the temporal profile of emitted two-photon fluorescence which is influenced by metabolic activity, redox ratio and other parameters30, 33, 35, 36. Changes in metabolism associated with cancer have been shown to alter the lifetimes of several common fluorescent cellular metabolites33. This proposal emphasizes the development of new technology for real-time imaging of cancer pathology. We will first develop a TPM system capable of imaging very large fields (>1 cm2) at high speed (>30 megapixels/s) using a fast optical scanning, an ultrashort pulse excitation and sample mosaicking (Aim 1). We will then develop an integrated system for high-speed (>1-10 megapixel/s) 3D TPM-FLIM whole tissue imaging using a novel optical sampling technique based on an optically clocked digitizer. In Aim 2, we propose to conduct a study of surgical specimens (discarded and not required for clinical diagnosis) in the pathology laboratory to develop both TPM and TPM-FLIM for real-time imaging and detection of breast and thyroid cancer pathologies. This study will identify biomarkers and sources of intrinsic contrast that can be used to detect cancer pathology. Finally the diagnostic performance of TPM-FLIM will be compared to the gold standard, histopathology in a blinded evaluation to determine sensitivity and specificity. If successful, this imaging technology could ultimately be applied to a range of intraoperative applications where real-time assessment of pathology is required.